Báo cáo y học: "Chotosan (Diaoteng San)-induced improvement of cognitive deficits in senescence-accelerated mouse (SAMP8) involves the amelioration of angiogenic/neurotrophic factors and neuroplasticity systems in the brain" pptx

RESEARC H Open Access
Chotosan (Diaoteng San)-induced improvement
of cognitive deficits in senescence-accelerated
mouse (SAMP8) involves the amelioration of
angiogenic/neurotrophic factors and
neuroplasticity systems in the brain
Qi Zhao
1,2
, Takako Yokozawa
2,3
, Koichi Tsuneyama
4
, Ken Tanaka
5
, Takeshi Miyata
6,7
, Notoshi Shibahara
2
and
Kinzo Matsumoto
1*
Abstract
Background: Chotosan (CTS, Diaoteng San), a Kampo medicine (ie Chinese medicine) formula, is reportedly
effective in the treatment of patients with cerebr al ischemic insults. This study aims to evaluate the therapeutic
potential of CTS in cognitive deficits and investigates the effects and molecular mechanism(s) of CTS on learning
and memory deficits and emotional abnormality in an animal aging model, namely 20-week-old senescence-
accelerated prone mice (SAMP8), with and without a transient ischemic insult (T2VO).
Methods: Age-matched senescence-resistant inbred strain mice (SAMR1) were used as control. SAMP8 received
T2VO (T2VO-SAMP8) or sham operation (sham-SAMP8) at day 0. These SAMP8 groups were administered CTS (750
mg/kg, p.o.) or water daily for three weeks from day 3.
Results: Compared with the control group, both sham-SAMP8 and T2VO-SAMP8 groups exhibited cognitive

deficits in the object discrimination and water maze tests and emotional abnormality in the elevated plus maze
test. T2VO significantly exacerbated spatial cognitive deficits of SAMP8 elucidated by the water maze test. CTS
administration ameliorated the cognitive deficits and emotional abnormality of sham- and T2VO-SAMP8 groups.
Western blotting and immunohistochemical studies revealed a marked decrease in the levels of phosphorylated
forms of neuroplasticity-related proteins, N-methyl-D-aspartate receptor 1 (NMDAR1), Ca
2+
/calmodulin-dependent
protein kinase II (CaMKII), cyclic AMP responsive element binding protein (CREB) and brain-derived neurotrophic
factor (BDNF) in the frontal cortices of sham-SAMP8 and T2VO-SAMP8. Moreover, these animal groups showed
significantly reduced levels of vasculogenesis/angiogenesis factors, vascular endothelial growth factor (VEGF), VEGF
receptor type 2 (VEGFR2), platelet-derived growth factor-A (PDGF-A) and PDGF receptor a (PDGFRa). CTS treatment
reversed the expression levels of these factors down-regulated in the brains of sham- and T2VO-SAMP8.
Conclusion: Recovery of impaired neuroplasticity system and VEGF/PDGF systems may play a role in the
ameliorative effects of CTS on cognitive dysfunction caused by aging and ischemic insult.
* Correspondence:
1
Division of Medicinal Pharmacology, Institute of Natural Medicine, University
of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
Full list of author information is available at the end of the article
Zhao et al. Chinese Medicine 2011, 6:33
/>© 2011 Zhao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the t erms of the Creative Comm ons
Attribution License (http://creativecommons.o rg/licenses/by/2.0), which p ermits unrestricted use, distribution, and reproduction in
any medium , provided the original work is properly cited.
Background
Chotosan (CTS, Diaoteng San)isaKampo(ie Chinese
medicine) formula consisting of ten medicinal herbs and
gypsum fibrosum. It has long been used to treat chr onic
headache and hy pertension, particularly in middle-aged
or older patients with weak physical constitutions,
chronic headache, painful tension of the shoulders and

cervical muscles, vertigo, morning headache, a heavy
feeling of the head, flushing, tinnitus, and insomnia [1].
In a double-blind and placebo-controlled clinical study
[1], CTS showed an ameliorative effect on cognitive dys-
functions in stroke patients. CTS and tacrine (a choli-
nesterase inhibitor) exhibit a preventive effect on
cognitive deficits in a mouse model of transient cerebral
ischemia and a ther apeutic effect on learning and mem-
ory impairments in a mo use model of chronic cerebral
hypoperfusion [2,3]. These findings suggest that CTS
may be used as an anti-dementia drug. However, since
the beneficial effects of CTS have been demonstrated in
young animals from eight to 15 weeks old, it is still
unclear whether CTS is applicable to treat cognitive dys-
function caused by ischemic insult in aged animals.
Aging is a risk factor for a variety of diseases including
deterioration of brain function [4]. One of the promi-
nent symptoms due to aging-induced brain dysfunction
is cognitive deficits such as in Alzheimer disease (AD)
and cerebrovascular disease-related dementia [5].
Indeed, the incidence rates of AD and cerebrovascular
dementia increase with aging [4,6]. It has been suggested
that cerebrovascular diseases also play an important role
in the pathogenic mechanism(s) underlying spo radic
(non-genetic) AD [4,7] and that patients with AD
pathology often have concomitant cerebrovascular
pathology [8,9]. In fact, aging causes impaired angiogen-
esis that is in part attributable to a decrease in angio-
genic growth factors such as VEGF [7]. Retardation of
angiogenesis in the brains of aged animals is severe

enough to impair synaptic plasticity, a molecular biolo-
gical process important in learning and memory, and
requires long-lasting increases in metabolic de mand
supported by the generation of new capillaries [10].
Moreover, recent evidence has shown that the VEGF
and platelet-derived growth factor (PDGF), angiogenic
growth factors are important not only in angiogenesis
but also in neuroprotection and neurogenesis in the
brain [11] and that elevation of these factors improves
cognitive deficits and mental activity in aged animals
[12-14]. Therefore, drugs used to treat cerebrovascular
dementia or drugs with a potential to affect angiogenic
factors are likely to be beneficial for cognitive dysfunc-
tions related to aging.
The senescence-accelerated mouse (SAM) is a model
of accelerated senescence established by phenotypic
selection from a common genetic pool of AKR/J strain
mice [15]. In particular, SAMP8 is one of the strains
that exhibit early development of a variety of aging-
related symptoms such as impaired immune responses,
cognitive deficits [13,16,17], emotional disorders [13,15]
andelevatedexpressionofamyloid precursor protein
and b-amyloid in the brain [18]. Evidence indicates that
cognitive deficits in SAMP8 can be observed as early as
four months after birth, which is earlie r than those in
SAMR1 and that the deficits appear to be due to dys-
function of the neurobiological signaling mediated by
some key proteins such as Ca
2+
/calmodulin-dependent

kinase II (CaMKII), cyclic AMP responsive element
binding protein (CREB) and N-methyl-D-aspartate
receptor (NMDAR), which are important for synaptic
plasticity [13,15,19,20]. Moreover, our previous study
suggested that the VEGF/VEGFR2 signaling system in
the brain is down-regulated in the SAMP8 animals and
that the amelioration of cognitive deficits of SAMP8
implies the improvement of the system [13]. These fea-
tures of SAMP8 provide a useful animal model for the
investigation of the neurological and molecular biologi-
cal basis for cognitive dysfunction caused by aging in
humans.
This study investigates the effect of CTS on cognitive
deficits in an animal aging model, namely SAMP8, with
and without ischemic insult, to evaluate whether CTS
can be used as an anti-dementia drug to treat aging-
related cognitive deficits.
Methods
Animals
Male SAMP8 and SAMR1 aged six weeks were obtained
from SLC Inc. (Japan). Mice were housed in a laboratory
animal room maintained at 25 ± 1°C with 65 ± 5%
humidity on a 12-hour light/dark cycle (07:30 to 19:30).
Animals were given food and water ad libitum. The pre-
sent study was conducted in accordance with the Guid-
ing Principles for the Care and Use of Animals (NIH
publication #85-23, revised in 1985) and complied with
the Helsinki Declaration [21]. The present study was
also approved by the Institutional Animal Use and Care
Committee of the University of Toyama. A detailed

experimental schedule is described in Figure 1.
Preparation and chemical profiling of CTS
CTS extract used in this study was purchased from Tsu-
mura Co. (Japan) in the form of a spray-dried powder
extract prepared according to the standardized extrac-
tion method of medicinal plants registered in the Japa-
nese Phar macopoeia XV. The CTS extract was from the
same lot (Lot #202004-7010) used in a previous study
[2]. This extract was prepared from a mixture of 3.0
parts Uncariae Uncis cum Ramulus ( hooks and branch
of Uncaria rhynchoph ylla MIQUEL), 3.0 parts Aurantii
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 2 of 18
Nobilis pericarpium (peel of Citrus unshiu MARKO-
VICH), 3.0 parts Pinelliae tuber (tuber of Pinellia ter-
nate BREITENBACH), 3.0 parts Ophiopogonis tuber
(root of Ophiopogon japonicus KER-GAWLER), 3.0
parts Hoelen (sclerotium of Poria cocos WOLF), 2.0
parts Ginseng radix (root of Panax ginseng C.A.
MEYER), 2.0 parts Saphoshnikoviae radix et rhizoma
(root and rhizome of Saposhnikovia divaricata
SCHISCHKIN), 2.0 parts Chrysanthemi flos (flower of
Chrysanthemum morifolium RAMATULLE), 1.0 part
Glycyrrhizae radix ( root of Glycyrrhiza uralensis
FISHER), 1.0 part Zingiberis rhizoma (rhizome of Zingi-
ber officinale ROSCOE) and 5.0 parts Gypsum fibrosum
(CaSO
4
2H
2

O). The yield of the CTS extract was 16.1%.
To identify the chemical constituents of CTS, 3D-
HPLC analysis was conducted as previously described
[2,3]. Briefly, CTS (2.5 g, Tsumura, Japan) was filtered
and then sub jected to high-performance liquid chroma-
tography (HPLC) analysis. HPLC equipment was con-
trolled by an SLC-10A system controller (Shimadzu,
Japan) with a TSKGELODS-80TS column (4.6×250 mm)
(TOSOH, Japan), eluting with solvents (A) 0.05 M
AcONH
4
(pH3.6) and (B) CH
3
CN. A linear gradient
(100% A and 0% B to 0% A and 100% B in 60 minutes)
was used. The flow rate was controlled by an LC-10AD
pump (Shimadzu, Japan) at 1.0 ml/min. The eluent from
the column was monitored and processed with an SPD-
M10A diode array detector (Shimadzu, Japan). The 3D-
HPLC profiling data have been previously described
[2,3]. For chemical profiling of CTS, liquid chromatogra-
phy-mass spectrometry (LC-MS) analysis was performed
with a Shimadzu LC-IT-TOF mass spectrometer (Japan)
equipped with an ESI interface (Shimadzu, Japan). The
ESI parameters were as follows: source voltage +4. 5 kV,
capillary temperature 200°C and nebulize r gas 1.5 l/min.
The mass spectrometer was operated in positive ion
mode scanning from m/z 200 to 2000. A Waters Atlan-
tis T
3

column (2.1 mm i.d. × 150 mm, 3 m, USA) was
used and the column temperature was maintained at 40°
C. The mobile phase was a binary eluent of (A) 5 mM
ammonium acetate solution and (B) CH
3
CN under the
following gradient conditions: 0-30 minutes linear gradi-
ent from 10% to 100% B, 30-40 min isocratic at 100% B.
The flow rate was 0.15 ml/min. Mass spect romet ry data
obtained from the extract were deposited in MassBank
database [22] and stored with the pharmacological infor-
mation on the extract in the Wakan-Yaku Database sys-
tem [23], Institute of Natural Medicine, University of
Toyama. The CTS extract used in this study was depos-
ited at our institute (voucher specimen no. 20000005).
Surgical operation for transient cerebral ischemia
Surgical operation to induce transient cerebral ischemia
(T2VO) was conducted as previously described [3].
Briefly, at the age of 20 weeks, SAMP8 received transi-
ent occlusion of bilateral common carotid arteries for 15
minutes under pentobarbital-Na (50 mg/kg, i.p.)
anesthesia. The animals that received the same opera-
tion without occlusion of carotid arteries served as
sham-operated controls. From three days after the
operation, the animals received daily administration of
CTS (750 mg/kg, p.o.). The dose of CTS was selected
on the basis of our previous studies using an animal
model of cerebrovascular dementia [2,3].
Behavioral assessment
Elevated plus maze test

The elevated plus maze is comprised of two open arms
(22 × 8 cm) and two arms enclosed by high walls (22 ×
8 × 17 cm), with an open roof, the two arms of each
type being positioned opposite to each other as pre-
viously described [13]. The maze was set 60 cm above
the floor. Each mouse was individually placed at the
center of the maze facing one of the encl osed arms and
allowedtoexplorethemazefreelyduringa5-minute
observation period. Maze performance was video-
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Figure 1 Schematic drawing of the experimental schedule in this study. Transient ischemic operation was conducted at day 0. From day 3,
administration of CTS to the SAMP8 group was started.
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 3 of 18
recorded for later analysis. Time spent in open arms and
the numbers of arm entries were analyzed as indices of
emotional behavior using SMART
®
ver. 2.5 (PanLab,
SLU, Spain).

Learning and memory test
Nobel object recognition test (ORT) ORT w as con-
ducted as previously described [2,13] with minor modifi-
cations. The appar atus consisted of a square arena (50 ×
50 × 40 cm) made of polyvinyl chloride with gray walls
and a black floor. The objects for recognition had visual
patterns or visual ly different shapes to be discriminated.
The ORT consisted of a sample phase trial and a test
phase trial. In the sample phase trial, each mouse was
first placed in the observation box where two identical
objects, namely O1 and O2 (each of which was a 7.5 ×
5.5 cm white cup), were placed separately and allowed
to freely explore the arena for five minutes. The total
time that the mouse spent exploring each of the two
objects was measured and then the mouse was returned
to the home cage. In the test phase trials performed ten
minutes after the sample phase trials, one of the two
objects was replaced by an identical copy (object F) and
the other by a novel object (object N). Performance of
the animals in this test was video-recorded for later ana-
lysis. In these trials, the exploration of an object was
defined as directing the nose to the object at a distance
of less than 2 cm according to previous reports [2,13]
and the time spent exploring each of the two objects
was analyzed with SMART
®
ver. 2.5 (PanLab, SLU,
Spain) with a tri-wise module to detect the head, center
mass and base-tail. A discrimination index (DI) was cal-
culated according to the following equation [2,13]:

DI =
(
T
n
− T
f
)
/
(
T
n
+T
f
)
where T
n
and T
f
represent the time spent exploring
new and familiar objects respectively. The box arena
and objects were clean ed with 75% ethanol between
trials to prevent a build-up of olfactory cues.
Nobel object location test (OLT) The OL T, which is a
two-trial task with a sample phase trial and a tes t phase
trial separated by an inter-trial interval, was conducted
as previously reported [2,13]. T he objects used in the
sample phase t rial were two black cones A1 and A2 (5
× 10 cm). Ten minutes after the sample phase trial, the
test phase trial was conducted. In this t rial, the objects
were replaced by their identical copies, one of which

was placed in the same position, whereas the other was
moved to the adjacent corner, so that the two objects
were in diagonally opposite co rners. In the test phase
trials, both objects were equally familiar to the animals,
but one had changed location. The mice were exposed
to the objects for five minutes. Performance of the ani-
mals was video-recorded and the total time spent
exploring each of the two objects was analyzed as pre-
viously described.
Morris water maze test The Morris water maze test
was conducted with a circular pool (110 cm in dia-
meter), a transparent platform (7 cm in diameter) and
various extra maze cues surrounding the pool as pre-
viously described [2,3]. Twenty-nine days after surgery,
the animals were subjected to a visible trial test (Visible
1) where the platform was made visible 1 cm above the
water surface. One day after the visible trial , acquisition
trials were performed daily for five days. The animals
underwent four trials daily. In each training trial, the
mouse was placed in the pool from one of the four start
positions at 90° apart around the edge of the pool and
then allowed to swim to the hidden transparent plat-
form (7 cm in diameter). If the mouse had not found
the platform during a 60 s period, it was placed onto
the platform by the experimenter. The mouse was
allowed to r emain on the platform for 10 s before being
placed in an opaque high-sided plastic chamber for 60 s.
The next trial was then performed. Water maze beha-
vior of each mouse was video-recorded for later analysis.
In each trial, the latency to reach the platform (escape

latency), distance covered and average swim speed wer e
analyzed via a video capture and image analysis system
(SMART
®
system, PanLab, SLU, Spain). The daily trial
data of each animal were averaged and expressed as a
block of four trials before statistical analysis. One day
after the last acquisition trials, a single 60 s probe trial
was run in which the platform was removed from the
pool. The time spent in each of the four imaginary
quadrants of the pool was recorded and analyzed with
the SMART
®
system.
Quantitative real-time PCR
Quantitative real-time PCR was conducte d as previously
described [13]. Briefly, the animals were decapitated
after completing the behavioral experiments. The c ere-
bral cortices w ere dissected out and kept at -80°C until
use. Total RNA was extracted from the cortex with
Sepazol
®
(Nacalai Tesque, Japan) according to the man-
ufacturer’s instructions. First-strand cDNA was synthe-
sized with oligo (dT) primers and M-MLV reverse
transcriptase
®
(Invitrogen, USA) and was used as a tem-
plate for real-time PCR. Quantitative real-time PCR was
carried out with Fast SYBR Green Master Mix and the

StepOne Real-time PCR System
®
(Applied BioSystem,
USA). The following primer sets of B DNF and b-actin
were designed by Perfect Real Time support system
(Takara Bio Inc., Japan): BDNF (NM_007540): 5’ -
AGCTGAGCTGTGTGACAGT-3’ (forward) and 5’ -
TCCATAGTAAGGGCCCGAAC-3’ (reverse); b-actin
(NM_007393): 5’ -CATCCGTAAAGACCTCTATGC-
CAAC-3’ (forward) and 5’ -ATGGAGCCACCGATCC
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 4 of 18
ACA-3’ (reverse ). Melting curve analysis of each gene
was performed every time after amplification. In all
reactions, b-actin mRNA was used as a control to which
the results were normalized. Standard curves of the log
concentration of each gene vs. cycle threshold were
plotted to prove inverse linear correlations. The co rrela-
tion coefficients for standard curves of target genes were
0.9965 to 0.998.
Western blotting analysis
Western blotting was performed as previously described
with minor modifi cations [13,24]. Briefly, tissu e samples
were taken from the cortices and homogenized in lysis
buffer TissueLyser
®
(Qiagen, Japan) consisting of 50
mM Tris HCl buffer (pH7.4), 150 mM NaCl, 0.5%
sodium deoxycholate, 1% (v/v) NP-40, 0.1% (v/v) sodium
dodecyl sulfate (SDS), 150 mM NaF, 8.12 μg/ml aproti-

nin, 2 mM sodium orthovanadate, 10 μg/ml leupeptin,
and 2 mM phenylmethylsulfonyl fluoride. The lysate
samples were then centrifuged at 10,000 rpm (9200 g,
Kubota 3740, Kubota Co., Japan) at 4°C for five minutes.
The protein concentration of the supernatant was deter-
mined with a BCA™ protein assay kit (Thermo Scienti-
fic, USA). Each protein sample was mixed with Laemmli
sample buffer and denatured at 95°C for three minutes.
The proteins (20 μg) from each sample were electro-
phoresed on 5-12% sodium dodecyl sulfate polyacryla-
mide gel (SDS-PAGE) and then electro-blotted onto a
polyvinylidene difluoride membrane (Bio-rad Laboratory,
USA). The membranes were incubated in a 5% non-fat
milk-containing wash buffer (Nacarai Tesque, Japan) (50
mM Tris HCl pH7.5, 150 mM NaCl and 0.1% Tween
20)foronehouratroomtemperature.Theywerethen
probed with anti-NMDAR1 rabbit polyclonal antibody
(1:1000 dilution) and anti-phospho-NMDAR1 (p-
NMDAR1) (Ser896) rabbit polyclonal antibody (1:1000
dilution), anti-CaMKIIa (A-1: sc-13141) mouse mono-
clonal antibody (1:1000 dilution), anti-phospho-CaMK II
(p-CaMKII) (Thr286) rabbit polyclonal antibody (1:1000
dilution) (Cell Signaling Technology, USA), anti-CREB
(48H2) rabbit monoclonal antibody (1:1000 dilution),
anti-phospho-CREB (p-CREB) (Ser133) rab bit monoclo-
nal antibody (1:1000 dilution), anti-BDNF (Tyr951) rab-
bit polyclonal antibody (1:500 dilution), anti-VEGF (A-
20: sc-152) rabbit polyclonal antibody (1:1000 dilution)
(Santa Cruz Biotechnology, USA), and anti-VEGFR2
(Ab-951) rabbit polyclonal antibody (1:1000 dilution)

(Signalway Antibody, USA) and anti-glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) mouse monoclonal
antibody (1:2000 dilution) (Chemicon, USA) at 4°C for
24 hours. After the membranes were rinsed in wash buf-
fer without non-fat milk, the blots were incubated with
anti-mouse or anti-rabbit secondary antibodies linked
with horseradish peroxidase (Dako Cytomation EnVision
+ System-HRP-labeled Polymer) (Dako Cytomation Inc.,
USA) according to the manufacturer’s instr uctions. The
quantity of immunoreactive bands was detected by an
enhanced chemiluminescence method (ImmobilonTM
Western Chemiluminescent HRP Substrate) (Millipore,
USA) and imaged with Lumino Image Analyzer LAS-
4000 (Fuji Film, Japan). The signal intensity was normal-
ized by comparing with their expression le vels in treat-
ment-naïve control mice. Each membrane was re-
probed with Blot Restore Membrane Rejuvenation Kit
(Chemicon, USA). The band images were analyzed with
VH-H1A5 software (Keyence, Japan).
Immunohistochemistry
CTS administration-induced changes in expression
levels of VEGF and P DGF-A in the cerebral cortex of
sham-SAMP8 and T2VO-SAMP8 were also examined
with immunohi stochemical analysis. Briefly, the animals
were fixed by intracardiac perfusion of 4% paraformalde-
hyde in phosphate buffered saline (PBS) under pento-
barbital anesthesia. Brains were post-fixed with 4%
paraformaldehydeovernightat4°C.Aseriesof5μm
coronal sections from different brain regions including
cerebral cortex and hippocampus were obtained. The

paraffin-embedded specimens were deparaffinized in
xylene and dehydra ted with ethanol. Endogenous perox-
idase was blocked with 0.1% hydrogen peroxide-metha-
nol for 30 minutes at room temperature. Washed with
Tris-buffered saline (TBS), the specimens were incu-
bated in a microwave oven (95°C, 750 W; MF-2; Nissin,
Japan) in target retrieval solution (Dako, Denmark) for
15 minutes and then washed with distilled water and
TBS. Nonspecific binding was blocked by treatment
with a special blocking reagent (Dako, Denmark) for 15
minutes. The specimens were challenged with 1:200
dilution of anti-VEGF or anti-PDGF-A antibody and
then incubated in a moist box at 4°C overnight. Washed
with TBS, the specimens were incubated with a pe roxi-
dase-conjugated anti-rabbit IgG polymer (Env ision-PO
for Rabbit; Dako, Denmark). After three washes in TBS,
a reaction product was detected with 3,3’-diaminobenzi-
dine tetrahydrochloride (0.25 mg/ml) and hydrogen per-
oxide solution (0.01%). Counter-stained wit h
hematoxylin, the sections were rinsed, dehydrated, and
covered. Also included in each staining run were nega-
tive controls in which the primary antibody was omitted.
The images were captured with a microscope (AX-80,
Olympus, Japan).
Statistical analysis
Statistical analysis of the data was conducted according
to Curran-Everett and Benos [25]. All data are expressed
as mean ± standard deviation (SD). Statistical analyses
of the behavioral data comprised paired and unpaired
Zhao et al. Chinese Medicine 2011, 6:33

/>Page 5 of 18
Student’ s t-tests, a two-way analysis of variance
(ANOVA), or two-way repeated measures ANOVA fol-
lowed by the Student-Newman-Keuls test, as appropri-
ate. The mRNA and protein expression levels were
evaluated with Student’st-testoratwo-wayanalysisof
variance (ANOVA) followed by the Student-Newman-
Keuls test. The analysis was conducted using SigmaStat
®
ver 3.5 (SYSTAT Software Inc., USA). Differences of P <
0.05 were considered significant.
Results
Behavioral studies
Effect of CTS on emotional disorder of sham- and T2VO-
SAMP8 in the elevated plus maze test
The elevated plus maze test was conducted to elucidate
the effect of CTS on emotional deficits of SAMP8 that
hadreceivedshamorT2VOoperation.Thesham-and
T2VO-SAMP8 treated with vehicle spent a significantly
longer time exploring the open arms than the SAMR1
controls (t = -5.468, df = 17, P < 0.001, t-test). The
administration of CTS (750 mg/kg per day, p.o.) to
sham- and T2VO-SAMP8 reduced the proportion of
time spent in open arms by these animal groups [F
drug
treatment
(1,34) = 76.639, P<0.001, two-way ANOVA].
No significant difference in the effect of CTS and T2VO
operation on total arm entries was observed between
sham- and T2VO-SAMP8 [F

drug treatment
(1,34) =
0.00021, P = 0.989 and F
operation
(1,34) = 1.851, P =
0.183, two-way ANOVA] (Figure 2).
CTS amelioration of non-spatial cognitive deficits of sham-
and T2VO-SAMP8 in ORT
The non-spatial cognitive performance of sham- and
T2VO-SAMP8 was elucidated by the ORT. The sample
phase trials of the ORT revealed no differences in total
time spent exploring two identical objects between
SAMR1 and sham-SAMP8 [t = 0.206, df = 18, P =
0.839]. Moreover, there was no significant interaction
between T2VO operation and CTS administration in
terms of performance of SAMP8 groups in the sample
phase trials [F
operation × C TS treatment
(1,36) = 0.285, P =
0.597, two-way ANOVA] (Figure 3A). However, in the
test phase trials, SAMR1 spent a significantly longer
time exploring a novel object than exploring a familiar
object [t = 9.05, df = 9, P < 0.001; paired t-test], indicat-
ing preference for the novelty. By contrast, sham- and
T2VO-SAMP8 showed no preference for the novel
object [sham-SAMP8: t = -1.263, df = 9, P =0.238]or
still spent a longer time exploring the familiar object
than the novel object [T2VO-SAMP8: t = -3.413, df = 9,
P = 0.008, paired t-test]. Treatment of sham- and
T2VO-SAMP8 with CTS (750 mg/kg/day, p.o.) normal-

ized novel object reco gnition behavior of these animal
groups which spent a significantly longer time on the
novel object than on the familiar object [CTS-treated
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Figure 2 Effects of CTS administration on elevated plus maze performance of SAMP8 with and without ischemic insult. The 20-week-
old SAMP8 received sham operation or transient occlusion of common carotid arteries (T2VO) for 15 minutes on day 0 and then received oral
administration of water (vehicle) or 750 mg/kg CTS once daily during an experimental period. The elevated plus maze test was conducted on
days 17 and 18 after ischemic operation. Each datum represents the mean ± SD (9-10 mice per group). The proportion of time spent in open
arms (A) and the number of total arm entries (B) were calculated. The data are expressed as the mean ± SD
###
P < 0.001 vs. vehicle-treated
SAMR1 group (t-test). ***P < 0.001 vs. vehicle-treated sham- or T2VO-SAMP8 groups (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 6 of 18
sham-SAMP8:t=4.094,df=9,P = 0.003; CTS-treated
T2VO-SAMP8: t = 4.136, df = 9, P =0.003,pairedt-
test] (Figure 3B). Analysis of the DI values also revealed
that the CTS administration improved the object recog-
nitiondeficitoftheSAMP8(shamandT2VO)group
[F
CTS treatment

(1,36) = 37.061, P <0.001,two-way
ANOVA] and that no significant interaction was
observed between T2VO operation and CTS treatment
in the performance of SAMP8 groups [F
operation × CTS
treatment
(1,36) = 0.0307, P = 0.862]. Moreover, compared
with SAMR1, the DI value of the vehicle-treated
SAMP8 was significantly decreased (t = 6.845, df = 18,
P < 0.001, t-test) (Figure 3C).
Effect of CTS on special cognitive performance in OLT and
water maze test
Object location test In the OLT, analysis of the sample
phase trials revealed no significant differences in the
total exploration time spent on identical objects between
SAMR1 and sham-SAMP8 [t = 0. 192, df = 18, P =0.85,
t-test ] or among SAMP8 groups [F
operation × CTS treat-
ment
(1,36) = 0.210, P = 0.650] (Figure 4A). In the test
phase trials, the SAMR1 and CTS-treated SAMP8
groups clearly showed a preference for an object placed
in a novel location compared with an object placed in a
familiar location [SAMR1: t = -10.803, df = 9, P<0.001;
CTS-treated sham-SAMP8: t = -5.806, df = 9, P < 0.001;
CTS-treated T2VO-SAMP8: t = -3.359, df = 9, P =
0.008, paired t-test]. By contrast, the sham-SAMP8 and
T2VO-SAMP8 groups treated with water vehicle were
unable to discriminate a novel location from a familiar
location or spent more time exploring the object placed

in a familiar location [sham-SAMP8: t = 0.985, df = 9, P
= 0.350; and T2VO-SAMP8: t = 3.109, df = 9, P =
0.013, paired t-test] (Figure 4B). Two-way ANOVA of
the DI among the SAMP8 groups revealed a significant
effect of CTS treatment [F
CTS treatment
(1,36) = 24.961, P
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Figure 3 Effects of CTS on object discrimination performance of SAMP8 with and without ischemic insult in the object re cognition
test (ORT). The object recognition test was conducted on days 19-21 after T2VO operation. Each datum represents the mean ± SD (ten mice
per group). (A) The data from the sample trials of the ORT. The animal was placed into the arena where two identical sample objects made of
glass (objects O1 and O2) were placed in two adjacent corners of the arena and was allowed to explore for five minutes. There was no
significant difference in performance in the sample phase trial among the groups. (B) The data from the test phase trials conducted ten minutes
after the sample phase trials. In the test phase trials, the time animals spent exploring a familiar object or a new object was measured during a
5-minute observation period. ***P < 0.001 and **P < 0.01 vs. the time spent exploring a familiar object (paired t-test). (C) Discrimination index
(DI) in the ORT. DI was calculated as described in the text.
###
P < 0.001 vs. vehicle-treated SAMR1 group (t-test). ***P < 0.001 vs. vehicle-treated
SAMP8 group (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 7 of 18
< 0.001]. CTS administration significantly reversed DI

values to the levels of the SAMP8 control group. More-
over, compared with SAMR1, the DI of the vehicle-trea-
ted SAMP8 was significantly decreased (t = 5.635, df =
18, P < 0.001, t-test) (Figure 4C).
Morris water maze test In order to test whether T2VO
exacerbates spatial memory deficits of SAMP8, we used
the water maze test based on a hippocampus-dependent
learning paradigm (Figure 5). Each animal group could
learn the lo cation of the submerged platform following
repeated daily training [F
training
(4,56) = 2 8.377, P <
0.001, two-way repeated measures ANOVA] but the
escape latency of the sham-SAMP8 vehicle control
group was significantly longer than that of SAMR1 con-
trol [F
animal × training
(4,56) = 2.921, P =0.029,two-way
repeated measures ANOVA]. Moreover, the T2VO-
SAMP8 mice displayed significantly longer latencies
than the sham-SAMP8 group to find a platform
[F
operation
(1,13) = 5.241, P = 0.039, two-way repeated
measures ANOVA] in the training trials. We also exam-
ined the effect of C TS on spatial cognitive performance
of the sham- and T2VO-SAMP8 in the water maze test.
Daily treatment of sham-SAMP8 and T2VO-SAMP8
mice with 750 mg/kg CTS resulted in a significant
decrease in e scape latencies of these animal groups

[sham-SAMP8: F
CTS treatme nt
(1,11) = 7.076, P =0.022;
T2VO-SAMP8: F
CTS treatment
(1,17) = 59.484, P < 0.001,
two-way repeated measures ANOVA] (Figure 5A).
Intheprobetestconductedonedayaftera5-day
training period, swimming time of the sham-SAMP8
control in the target quadrant where the platform was
placed during training was significantly shorter than
that of the SAMR1 [t = 3.009, df = 14, P = 0.009, t-test].
The sham- and T2VO-SAMP8 groups treated with daily
administration of CTS (750 mg/kg) spent a longer time
swimming in the target quadrant than those of the
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Figure 4 Effect s of CTS on object discrimination performance of SAMP8 with and without ischemic insult in the object location test
(OLT). The OLT was conducted on days 23-25 after T2VO operation. Each datum represents the mean ± SD (ten mice per group). (A) The data
from the sample trials of the OLT. The animal was placed into the arena where two identical sample objects made of glass (objects A1 and A2)
were placed in two adjacent corners of the arena and was allowed to explore for five minutes. There was no significant difference in
performance in the sample phase trial among the groups. (B) The data from the test phase trials conducted ten minutes after the sample phase
trials. In the test phase trials, the time animals spent exploring an object placed in the familiar and a new location was measured during a 5-
minute observation period. ***P < 0.001 and **P < 0.01 vs. the time spent exploring the familiar location (paired t-test). (C) Discrimination index
(DI) in the OLT. DI was calculated as described in the text.
###
P < 0.001 vs. vehicle-treated SAMR1 group (t-test). ***P < 0.001 vs. vehicle-treated
sham- or T2VO-SAMP8 groups (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33

/>Page 8 of 18
vehicle-treated sham- and T2VO-SAMP8 groups [F
CTS
treatment
(1,28) = 8.599, P = 0.007, two-way ANOVA] but
no significant interaction was detected between the
T2VO operation and CTS treatment in the SAMP8
groups [F
operation × CTS treatment
(1,28) = 0.502, P =0.484,
two-way ANOVA] (Figure 5B).
Neurochemical studies
CTS reverses synaptic plasticity-related signaling down-
regulated in the cerebral cortex of sham- and T2VO-SAMP8
In order to understand the molecular mechanism(s)
underlying CTS-induced improvement of cognitive defi-
cits in the sham- and T2VO-SAMP8, we examined the
effects of CTS on synaptic plasticity-related signaling by
measuring phosphorylation activities of NMDAR1, CaM-
KII and CREB phosphorylation in the cortex areas (Fig-
ure 6). Compared with SAMR1, the sham-SAMP8
groups had significantly reduced levels of p-NMDAR1 [t
= 2.643, df = 8, P = 0.030, t-test], p-CaMKII [t = 2.746, df
=8,P = 0.025, t-test] and p-CREB (t = 4.677, df = 8, P =
0.002, t-test). CTS administration to the sham-SAMP8
and -T2VO groups significantly reversed the decreased
levels of p-NMDA [F
CTS treatment
(1,16) = 14.326, P =
0.002, two-way ANOVA], p-CaMKII [F

CTS treatment
(1,16)
= 15.952, P = 0.001, two-way ANOVA] and p-CREB
[F
CTS treatment
(1,16) = 11.262, P =0.004,two-way
ANOVA] in these animal groups. However, no significant
difference in the expression levels of NMDAR1, CaM-
KIIa and CREB was observed between the SAMR1 and
sham-SAMP8 or among the SAMP8 groups.
We also measured the expression levels of BDNF gene
transcript and BDNF protein which is a functio nal
molecule downstream of the transcriptional activity of
CREB, via CREB phosphorylation, in the brain (Figure
7). In contrast to the SAMR1, the SAMP8 had signifi-
cantly reduced levels of BDNF mRNA (t = 3.238, df = 8,
P = 0.012, t-test) and its protein (t = 3.964, df = 8, P =
0.011, t-test) in the cerebral cortex. Howev er, daily
admini stration of CTS to sham- and T2VO-SAMP8 sig-
nificantly reversed the decreases in the expression levels
of BDNF mRNA [F
CTS treatment
(1,16) = 19.746, P <
0.001, two-way ANOVA] and BDNF protein [F
CTS treat-
ment
(1,16) = 5.135, P = 0.038, two-way ANOVA] in
these animal groups (Figure 8). The extent to which
CTS reversed the expression level of the BDNF mRNA
was not significantly different between the sham- and

T2VO-SAMP8 groups. Western blotting analysis also
confirmed that the amelioration of the transcription
process of BDNF mRNA in sham- and T2VO-SAMP8
animals occurred after the daily CTS administration.
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Figure 5 CTS administration-induced amelioration of impaired water maze performance of SAMP8 mice with and wit hout ischemic
insult. The water maze test was conducted on days 29-40 after T2VO operation. (A) Learning performance of the animals elucidated in the
training test. Each data point indicates the mean escape latency ± SD for 6-10 animals in each group.
###
P < 0.001, *P < 0.05, and ***P < 0.001
(two-way ANOVA for repeated measurement). (B) Memory retrieval performance elucidated in the probe test. The test was conducted 24 hours
after the last training trials. Each datum represents the mean of time spent in the target quadrant ± SD
##
P < 0.01 compared with vehicle-
treated SAMR1 group. **P < 0.01 compared with respective vehicle-treated sham- or T2VO-SAMP8 group (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33

/>Page 9 of 18
Effects of CTS on the expression levels of VEGF and PDGF,
angiogenic and neurotrophic factors, in the cerebral cortex
of sham- and T2VO-SAMP
Since the VEGF/PDGF family has an giogenic and neuro-
trophic roles in the central nervous system and its level
declines with aging [26-28], we evaluated the effects o f
the CTS treatment on the VEGF/VEGF R2 and PDGF-A/
PDGFRa systems in the cerebral cortex. Western blotting
analysis (Figure 9) revealed that, compared with SAMR1,
the sham-SAMP8 groups showed reduced levels of VEGF
(t = 2.829, df = 8, P = 0.022, t-test), VEGFR2 (t = 2.328,
df = 8, P = 0.048, t-test), PDGF-A (t = 3.41, df = 8, P =
0.009, t-test) and PDGFR-a (t = 5.419, df = 8, P < 0.001,
t-test). However, t he CTS administration significantly
up-regulated the expression levels of VEGF [F
drug
(1,16)
= 16.008, P =0.001,two-wayANOVA],VEGFR2[F
CTS
treatment
(1,16) = 35.591, P < 0.001, two-way ANOVA],
PDGF-A [F
drug
(1,16) = 15.118, P = 0.001, two-way
ANOVA] and PDGFRa [F
CTS trea tment
(1,16) = 26.571, P <
0.001, two-way ANOVA] in the sham- and T2VO-
SAMP8 groups. No significa nt interaction between the

ischemic operation and CTS administration was observed
[VEGF: F
operation×CTS treatment
(1,16) = 0.244, P =0.628;
VEGFR2: F
operation×CTS treatment
(1,16) = 0.885, P =0.361;
PDGF-A: F
operation×CTS treatment
(1,16) = 0.0713, P =
0.793; PDGFRa:F
operation×CTS treatment
(1,16) = 0.0576, P
= 0.813]. Immunohistochemical experiments conducted
in this study (Figure 10) also revealed that the cortical
expression levels of VEGF and PDGF-A in the vehicle-
treated sham- and T2VO-SAMP8 groups were clearly
lower than those in the vehicle-treated SAMR1 and that
the CTS-treated SAMP8 groups had expression levels of
these factors comparable to those in the vehicle-treated
SAMR1 group.
Discussion
This study aimed to clarify whether CTS has the thera-
peutic potential for aging-related cognitive deficits. To
this end, we investigated the effects of CTS on emo-
tional and cognitive deficits in an animal model of
aging, namely SAMP8, with and without ischemic insult.
The results have demonstrated that daily administration
of CTS ameliorates both emotional and cognitive defi-
cits of SAMP8 with and without ischemic insult and

suggested that the effect on the deficits is attributable to
the recovery of neuroplasticity-related neuronal signal-
ing and the VEGF/PDGF signaling systems deteriorated
by aging.
CTS-induced improvement of emotional deficits of SAMP8
The elevated plus maze test conducted in this study
revealed that sham- and T2VO-SAMP8 groups
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Figure 6 Effec ts of CTS on expression levels of p-NMDAR1, NMDAR1, p-CaMKII, CaMKI Ia, p-CREB, CREB, and GAPDH in the cerebral
cortex of SAMP8 with and without ischemic insult. Typical photos indicating the expression levels of each factor in the cerebral cortex of
vehicle-treated SAMR1 control (lane 1), vehicle-treated sham-SAMP8 (lane 2), CTS (750 mg/kg/day)-treated sham-SAMP8 (lane 3), vehicle-treated
T2VO-SAMP8 (lane 4), and CTS-treated T2VO-SAMP8 group (lane 5). After completing the behavioral studies, the animals were decapitated and
proteins were extracted from the cerebral cortices in each animal group.
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 10 of 18

exhibited significantly reduced anxiety-like behavior
compared with the control animal group, namely
SAMR1, and that this abnormal emotional behavior was
attenuated by CTS. The reduced anxiety level observed
in SAMP8 animals matches t he previous findings
reported by Miyamoto et al. [29] and our group [13].
This characteristic behavioral symptom observed in
SAMP8 animals provides potential utility of this animal
strain as a model to investigate aging-related symptoms
of patients with dementia [29]. Indeed, it has been
reported that the emotional deficit of SAMP8 occurs in
an aging-related manner and is, to some extent, relevant
to dysfunction of central noradrenergic function in this
animal strain and that long-term and continuous infu-
sion of thyrotropin-rele asing hormone (TRH) appears to
be useful for treatment of the emotional disorders and
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Figure 7 Quantitative comparisons of CTS-induced changes in expression levels of p-NMDAR1, NMDAR1, p-CaMKII, CaMKIIa, p-CREB,
CREB, and GAPDH in the cerebral cortex of SAMP8 with and without ischemic insult. The data are expressed as the percentage of the
value obtained from naïve control SAMR1 mice. Each data column represents the mean ± SD obtained from 4-5 brain samples.
#
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##
P
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group (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 11 of 18
memory disturbance caused in SAMP8 [29]. Therefore,
it is of interest to note in this study that CTS adminis-
tration during an experimental period could ameliorate
emotional deficits of SAMP animals with and without
ischemic insult. Taken together with aforementioned
previous findings, the present results allow us to specu-
late that CTS administration improves emotional and
cognitive deficits under a mechanism similar to that of
TRH. The exact mechanism underlying the effe ct of

CTS on emotional deficits of SAMP8 requires further
investigation.
Ameliorative effects of CTS on cognitive deficits caused
by aging with ischemic factor
Cognitive deficit is one of the prominent symptoms
caused by deterioration of brain function [13,19] . More-
over, aging and ischemic insults are risk factors impli-
cated in the pathophysiology of cognitive deficits in
patients with dementia. Therefore, in this study, we first
evaluated the cognitive performance of SAMP8 mice
with and without ischemic insult to create an animal
model of dementia involving these risk factors. The
results have demonstrated that SAMP8 animals with
and without T2VO exhibit severely impaired learning
and memory performance in the tests used to evaluate
non-spatial (ORT) and spatial cognitive performance
(OLT and water maze test). These findings agree with
previous studies using the same strain of mice [13,30]
and a mouse model of cerebrovascular dementia
[2,3,31]. Moreover, the present findings have also
revea led that, altho ugh T2VO has no effect on impaired
short-term memory performance of SAMP8 in the ORT
or OLT, it significantly exacerbates spatial reference
memory performance. The reason for thi s different sus-
ceptibility of cognitive performances of SAMP8 to
T2VO operation between the object recognition-type
tests (ORT and OLT) and the water maze test is
unclear. It may be due to a difference in difficulty to
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Figure 8 Effects of CTS on BDNF mRNA and protein expression in the cerebral cortex. After completing the behavioral studies, we
decapitated the animals and prepared the protein and total RNA from the cerebral cortices. Real-time PCR analysis of BDNF mRNA (A) and
BDNF protein (B) expression in vehicle-treated SAMR1 (lane 1) and vehicle-treated sham-SAMP8 (lane 2), CTS (750 mg/kg/day)-treated sham-
SAMP8 (lane 3), vehicle-treated T2VO-SAMP8 (lane 4), and CTS-treated T2VO-SAMP8 (lane 5). Each data column represents the mean ± SD
obtained from 5 brain samples.
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P < 0.05 vs. vehicle-treated SAMR1 group (t-test). *P < 0.05 and ***P < 0.001 vs. respective vehicle sham- or
T2VO-SAMP8group (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 12 of 18
learn these tasks and/or to the extent to which an aging
factor of SAMP8 affects cognitive ability used in these
tests. Nevertheless, it is conceivable that SAMP8 with
T2VO can also be used as an appropriate model for
aging-related dementia.
It should be noted that CTS administration amelio-
rates not only emotional disorders but also learning
and memory deficits observed in SAMP8 animals with
and without ischemic insult. We have previously
reported that CTS treatment or the cholinesterase
inhibitor tacrine improves spatial and non-spatial cog-
nitive deficits caused by chronic cerebral hypoperfu-
sion in mice [2,31], indicating a therapeutic potential
of CTS for cerebrovascular dementia. Taken together,
the present results indicate that CTS administration
exhibits a beneficial effect on cognitive deficits attribu-
table not only to cerebrovascular impairments but also
to an aging factor, providing further pharmacological
evidence for the utility of CTS as a potential anti-

dementia drug.
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Figure 9 Effects of CTS on VEGF/VEGFR2 and PDGF-A/PDGFRa expression in the cerebral cortex of SAMP8 with and without ischemic
insult. After completing the behavioral studies, we decapitated the animals and extracted the total protein from the cerebral cortices as
described in the text. (A) Typical photos indicating the expression levels of VEGF, VEGFR2, PDGF, and PDGFRa in the cerebral cortex of vehicle-
treated SAMR1 control (lane 1), vehicle-treated sham-SAMP8 (lane 2), CTS (750 mg/kg/day)-treated sham-SAMP8 (lane 3), vehicle-treated T2VO-

SAMP8 (lane 4), and CTS-treated T2VO-SAMP8 group (lane 5). (B) Quantitative comparisons of each factor among different animal groups were
conducted as described in the text. The data are expressed as the percentage of the value obtained from naïve control SAMR1 mice. Each data
column represents the mean ± SD obtained from five brain samples.
#
P < 0.05,
##
P < 0.01 and
###
P < 0.001 vs. vehicle-treated SAMR1 group (t-
test). ***P < 0.001 vs. respective vehicle-treated sham- or T2VO-SAMP8group (two-way ANOVA).
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 13 of 18
Improvement of neuroplasticity-related signaling by CTS
administration
To obtain evidence at the molecular level for CTS-
induced improvement of cognitive deficits in SAMP8
with and without ischemic insult, we analyzed an
important molecular biological feature of learning and
memory in the brain, namely the expression of signaling
proteins relevant to neuroplasticity. Lines of evidence
have demonstrated that glutamatergic systems, such as
NMDAR, are one of the molecular bases underlying
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Figure 10 Immunohistochemical evaluation of the effect of CTS on aging-induced decrease in VEGF (A) and PDGF-A (B) in the
prefrontal cortex region. (1) vehicle-treated SAMR1, (2) vehicle-treated sham-SAMP8, (3) CTS (750 mg/kg/day)-treated sham-SAMP8, (4) vehicle-
treated T2VO-SAMP8 and (5) CTS-treated T2VO-SAMP8 group. Scale bars in each photo and inset represent 100 μm and 50 μm respectively.
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 14 of 18
learning and memory [32] and that phosphorylation of
some key proteins s uch as NMDAR, CAMKII and
CREB triggered by glutamate receptor stimulation is a
molecular mechanism underlying neuroplasticity [19].
Indeed, activation of NMDA-type glutamate receptors
increases the intra cellular Ca
2+
level via the glutamate-
gated Ca
2+
/Na
+
channels on neuronal membranes,
thereby activating calmodulin and other Ca
2+
-dependent
enzymes. This signaling elicits the phosphorylation of an
NMDAR (GluR1) subunit protein at Ser896 in the hip-
pocampal and cortical glutamatergic synapses via the
activat ion of protein kinase C. Moreover, autophosphor-
ylation of CaMKII triggered by intracellular Ca

2+
-depen-
dent activation of calmodulin is reportedly implicated in
the conversion of short-term memory to long-term
memory. This process appears to potentiate cognitive
functi on by affecting a BDN F system [33]. CREB, one of
the nuclear transcription factors targeted by protein
kinases A and/or C, also plays an important role in
memoryformationinavarietyofcognitivetasksinvol-
ving different brain structures [34]. Its phosphorylated
form, p-CREB, is implicated in the transcription of late
downstream genes encoding proteins essential for learn-
ing and memory such as structural proteins, signaling
enzymes o r neurotrophic/growth factors including
BDNF [5]. In the present study, we analyzed these fac-
tors in the cortex since the cortex plays an important
role in object recognition performance [35]. As shown
in Figures 6 and 7, the vehicle-treated sham- and
T2VO-SAMP8 groups had significantly reduced levels of
p-NMDAR1, p-CaMKII, and p-CREB in the cortex com-
pared with the age-matched SAMR1 cont rol. In addi-
tion, expression levels of BDNF mRNA and its protein
were decreased in these animal groups. These findings
are consistent with our previous report [13]. Together,
our findings indicate that dysfunction of signal transduc-
tion mechanisms related to memory formations is also
caused in SAMP8, supporting the idea that the reduc-
tion of these phosphorylated proteins is linked to and/or
represents the impaired performance of SAMP8 in the
memory tasks.

Importantly, daily administration of CTS significantly
reversed an aging-induced decrease in phosphorylation
of NMDAR1, CaMKII, and CREB, as well as the expres-
sion of BDNF mRNA and its protein in the cerebral cor-
tex. The detailed mechanism underlying these actions of
CTS in SAMP8 groups is unclear. However, considering
thecloselinkageofthesefactorstotheBDNFprotein
and mRNA expressions in the brain, it is likely that the
ameliorative effects of CTS on cognitive deficits caused
in SAMP8 with and without ischemic insult are in part
due to the improvement of neurona l signaling mediated
by the glutamate receptor including an NMDAR
subtype.
We previously reported on res ults of a study using an
animal model of cereb rovascular dementia that the anti-
dementia effect of CTS is in part mediated by the
enhancement of central cholinergic function, particularly
M
1
muscarinic receptor stimulation [2,3,31]. Together,
the present findings raise the possibility that CTS-
induced reversal of impaired neurosignaling system in
the brain of the SAMP8 groups is one of the outcomes
resulting from enhancement of central cholinergic
mechanisms by CTS. This idea is supported by lines of
evidence. Endogenous acetylcholine reportedly exhibits a
facilitatory role in the NMDA receptor functio n via M1
muscarinic receptors in the brain [36]. Moreover, evi-
dence shows that the M
1

receptor-mediated cognition
behavior is mediated by neurosignaling pathways includ-
ing the CREB phosphorylation and BDNF expression
[37].
It has been demonstrated that aging process leads to
an imbalance between oxidative damage and antioxida-
tive defense system, and that most aging-induced phy-
siological changes are due to molecular and cellular
damage caused by free radicals [38]. In fact, reactive
oxygen species (ROS) play a role in many neurodegen-
erative diseases including Alzheimer’s disease [5,39,40]
and ROS are accumulated in the blood and brain in the
aging process, leading to the learning and memory defi-
cits observed in SAMP8 [40]. Therefore, it is likely that,
in this study, CTS administration attenuated the ele-
vated level of oxidative stress in the brain, thereby sup-
pressing cognitive impairment caused by oxidative brain
damage in SAMP 8. This possibility seems plausible
because previous reports from our laboratory demon-
strated that CTS effectively inhibited an oxidative stress-
related process via enhancing antioxidant enzyme activ-
ity and scavenging ROS [41].
A possible role of the VEGF and PDGF systems in the
anti-dementia effects of CTS in SAMP8
One significant finding of this study is that SAMP8
groups with and without ischemic insult exhibited
down-regulation of the VEGF/PDGF signaling system in
the brain and that the downregulation could be reversed
by CTS administration. VEGF is a hypoxia-inducible
secreted protein that interacts with receptor tyrosine

kinases such as VEGFR2 on endothelial cells, thereby
promoting angiogenesis. Aging is known to cause
impairment of angiogenesis via an alteration in extracel-
lular matrix and a reduction of angiogenic growth fac-
tors such as VEGF [7,9]. In the central nervous system
(CNS), VEGF and VEGFR2 are widely expressed not
only in vascular endothelial cells but also in neurons,
astrocytes, and neural progenitor cells [40]. Moreover,
VEGF exerts pleiotropic effects on brain functions
including enhancement of adult neurogenesis through
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 15 of 18
the direct activation of neural progenitor cells [42] and
ameliorates cognitive deficits via the promotion of neu-
rogenesis and its action as a protective factor for
endothelial cells and neurons during brain ischemia in
adu lt rats [42,43]. In fact, retar dation of angiogenesis in
the brain appears to become severe enough in aged ani-
mals to impair the neuroplasticity processes since neu-
roplasticity requires long-lasting increases in metabolic
demand supported by the generation of new capillaries
[10]. In addition, a recent study by Kim et al.[44]
revealed that exogenously applied VEGF elevates intra-
cellular Ca
2+
, activates CaMKII and potentiates long-
term potentiation in the hippocampal neurons. In this
study, we found that t he expressions of VEGF and
VEGFR2 and their genes were decreased in the brains of
older SAMP8, indicating aging-induced dysfunction of

the VEGF-VEGFR2 signaling system. Therefore, the
impai red VEGF-VEGFR 2 signaling systems likely induce
a decrease in angiogenesis and neuronal signaling in the
brain and are implicated in aging-induced cognitive defi-
cits in SAMP8. Considering a role of the VEGF-
VEGFR2 signaling systems in cognitive function, we, in
this study, raise the possibility that the CTS-induced
reversal of the impaired VEGF-VEGFR2 signaling system
is also a part of the mechanism(s) underlying the ameli-
orative effects of CTS on spatial and non-spatial cogni-
tive deficits caused by aging.
It is of interest to note from the present neurochem-
ical and immunohistochemical studies that the PDGF/
PDGFRa system as well as the VEGF/VEGFR2 system
was also down-regulated in the brain of the sham- and
T2VO-SAMP8 groups compared with that in the con-
trol group SAMR1 and that the down-regulation was
reversed by the CTS administration in both SAMP8
groups. These findings raise the possibility that aging
reduces the function of the PDGF/PDGFR signaling
system and that recovery of this system may play a
role in the CTS-induced amelioration of cognitive defi-
cits caused by aging with and without ischemic insult.
This possibility i s supported by a couple of factors.
Firstly, evidence indicates that PDGF-A and -B and
their receptors (PDGFRa and PDGFRb) expressed in
the CNS [45] are implicated not only in the prolifera-
tion, migration and differentiation of oligodendrocytes
[46] but also in neurite outgrowth [47], positive and
negative modulations of NMDA-receptor function via

the PKC and PKA pathways respectively [48] and neu-
roprotection via phosphatidylinositol 3-kinase, a mito-
gen-activated kinase pa thway [28]. These signaling
mechanisms are important in the long-term potentia-
tion of learning and memory, a biological index of
memory formation [49]. Recently, it was reported that
PDGFRa and PDGFb are implicated in the conversion
of oligodendroglia progenitor cells to pyramidal
neurons in adult piriform cortex [50] and induct ion of
neuroplasticity [51] respectively. Secondly, expression
levels of the PDGFs and PDGF receptors and the acti-
vation of mitogen-activated protein kinases via a
PDGF/PDGFR signaling pathway are down-regulated
by aging [26]. Taken together with the proposed roles
of the PDGF/PDGFR systems in the brain, it is plausi-
ble that the CTS-induced reversal of expression levels
of PDGF and PDGFR in SAMP8 with and without
ischemic insult contributes to the improvement o f cog-
nitive performance by CTS administration.
Conclusion
CTS can ameliorate emotional abnormality and cogni-
tive deficits caused by aging factors including ischemic
insults and the recovery of an impaired neuroplasticity
system and VEGF/PDGF systems plays an important
role in the ameliorative effects of CTS on cognitive dys-
function. CTS is a potential therapeutic agent for aging-
related cognitive dysfunctions.
Abbreviations
CTS: Chotosan; SAMP8: senescence-accelerated prone mice 8; SAMR1:
senescence-resistant inbred strain mice; T2VO: transient two vessel occlusion;

NMDAR1: N-methyl-D-aspa rtate receptor 1; CaMKII: Ca
2+
/calmodulin-
dependent protein kinase II; CREB: cyclic AMP responsive element binding
protein; BDNF: brain-derived neurotrophic factor; VEGF: vascular endothelial
growth factor; VEGFR2: VEGF receptor type 2; PDGF-A: platelet-derived
growth factor-A; PDGFRα: PDGF receptor α; AD: Alzheimer disease; 3D-HPLC:
three dimensional high performance liquid chromatography; ORT: object
recognition test; OLT: object location test; PCR: polymerase chain reaction;
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel; GAPDH:
glyceraldehyde-3-phosphate dehydrogenase; TBS: Tris-buffered saline;
ANOVA: analysis of variance.
Acknowledgements
This work was in part supported by a grant-in-aid (B) (#20390197) to KM.
Author details
1
Division of Medicinal Pharmacology, Institute of Natural Medicine, University
of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
2
Division of Kampo
Diagnostics, Institute of Natural Medicine, University of Toyama, 2630
Sugitani, Toyama 930-0194, Japan.
3
Collaboration Division, Organization for
Promotion of Regional Collaboration, University of Toyama, 3190 Gofuku,
Toyama 930-8555, Japan.
4
Department of Diagnostic Pathology, Graduate
School of Medical and Pharmaceutical Sciences, University of Toyama, 2630
Sugitani, Toyama 930-0194, Japan.

5
Division of Pharmacognosy, Institute of
Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194,
Japan.
6
Division of Biomedical Informatics, Institute of Natural Medicine,
University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
7
Laboratory of
Presymptomatic Medical Pharmacology, Faculty of Pharmaceutical Sciences,
Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.
Authors’ contributions
KM designed the study and wrote the manuscript. QZ conducted the
behavioral and neurochemical studies. TY participated in the design of the
study using SAMP8 and SAMR1. KOT designed the immunohistochemical
study and helped analyze the data. KET conducted chemical profiling of the
extract. TM conceived the study and helped draft the manuscript. NS
participated in the study design and helped draft the manuscript. All authors
read and approved the final version of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Zhao et al. Chinese Medicine 2011, 6:33
/>Page 16 of 18
Received: 17 June 2011 Accepted: 23 September 2011
Published: 23 September 2011
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doi:10.1186/1749-8546-6-33
Cite this article as: Zhao et al.: Chotosan (Diaoteng San)-induced
improvement of cognitive deficits in senescence-accelerated mouse
(SAMP8) involves the amelioration of angiogenic/neurotrophic factors
and neuroplasticity systems in the brain. Chinese Medicine 2011 6:33.

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